Technetium-99m labeled peptides for imaging

ABSTRACT

This invention relates to radiolabeled peptides and methods for producing such peptides. Specifically, the invention relates to peptides, methods and kits for making such peptides, and methods for using such peptides to image sites in a mammalian body labeled with technetium-99m (Tc-99m) via a radiolabel-binding moiety which forms a neutral complex with Tc-99m.

This application claims priority to International ApplicationPCT/US93/03687, filed Apr. 19, 1993, which is a continuation-in-part ofU.S. Ser. No. 07/871,282, filed Apr. 30, 1992, incorporated by referenceherein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to radiodiagnostic reagents and peptides, andmethods for producing labeled radiodiagnostic agents. Specifically, theinvention relates to peptides, methods and kits for making suchpeptides, and methods for using such peptides to image sites in amammalian body labeled with technetium-99m (Tc-99m) via aradiolabel-binding moiety which forms a neutral complex with Tc-99m.

2. Description of the Prior Art

In the field of nuclear medicine, certain pathological conditions arelocalized, or their extent is assessed, by detecting the distribution ofsmall quantities of internally-administered radioactively labeled tracercompounds (called radiotracers or radiopharmaceuticals). Methods fordetecting these radiopharmaceuticals are known generally as imaging orradioimaging methods.

In radioimaging, the radiolabel is a gamma-radiation emittingradionuclide and the radiotracer is located using a gamma-radiationdetecting camera (this process is often referred to as gammascintigraphy). The imaged site is detectable because the radiotracer ischosen either to localize at a pathological site (termed positivecontrast) or, alternatively, the radiotracer is chosen specifically notto localize at such pathological sites (termed negative contrast).

A number of factors must be considered for optimal radioimaging inhumans. To maximize the efficiency of detection, a radionuclide thatemits gamma energy in the 100 to 200 keV range is preferred. To minimizethe absorbed radiation dose to the patient, the physical half-life ofthe radionuclide should be as short as the imaging procedure will allow.To allow for examinations to be performed on any day and at any time ofthe day, it is advantageous to have a source of the radionuclide alwaysavailable at the clinical site.

A variety of radionuclides are known to be useful for radioimaging,including ⁶⁷ Ga, ^(99m) Tc (Tc-99m), ¹¹¹ In, ¹²³ I, ¹²⁵ I, ¹⁶⁹ Yb, or¹⁸⁶ Re. Tc-99m is a preferred radionuclide because it emits gammaradiation at 140 keV, it has a physical half-life of 6 hours, and it isreadily available on-site using a molybdenum-99/technetium-99mgenerator.

The sensitivity of imaging methods using radioactively-labeled peptidesis much higher than other radiopharmaceuticals known in the art, sincethe specific binding of the radioactive peptide concentrates theradioactive signal over the area of interest. Small synthetic peptidesthat bind specifically to targets of interest may be advantageously usedas the basis for radiotracers. This is because: 1. they may besynthesized chemically (as opposed to requiring their production in abiological system such as bacteria or mammalian cells, or theirisolation from a biologically-derived substance such as a fragment of aprotein); 2. they are small, hence non-target bound radiotracer israpidly eliminated from the body, thereby reducing background(non-target) radioactivity and allowing good definition of the target;and 3. small peptides may be readily manipulated chemically to optimizetheir affinity for a particular binding site.

Small readily synthesized labeled peptide molecules are preferred asroutinely-used radiopharmaceuticals. There is clearly a need for smallsynthetic labeled peptides that can be directly injected into a patientand will image pathological sites by localizing at such sites. Tc-99mlabeled small synthetic peptides offer clear advantages as radiotracersfor gamma scintigraphy, due to the properties of Tc-99m as aradionuclide for imaging and the utility of specific-binding smallsynthetic peptides as radiotracer molecules.

Radiolabeled peptides have been reported in the prior art.

Ege et al., U.S. Pat. No. 4,832,940 teach radiolabeled peptides forimaging localized T-lymphocytes.

Olexa et al., 1982, European Patent Application No. 823017009 disclose apharmaceutically acceptable radiolabeled peptide selected from FragmentE₁ isolated from cross-linked fibrin, Fragment E₂ isolated fromcross-linked fibrin, and peptides having an amino acid sequenceintermediate between Fragments E₁ and E₂.

Ranby et al., 1988, PCT/US88/02276 disclose a method for detectingfibrin deposits in an animal comprising covalently binding aradiolabeled compound to fibrin.

Hadley et al., 1988, PCT/US88/03318 disclose a method for detecting afibrin-platelet clot in vivo comprising the steps of (a) administeringto a patient a labeled attenuated thrombolytic protein, wherein thelabel is selectively attached to a portion of the thrombolytic proteinother than the fibrin binding domain; and (b) detecting the pattern ofdistribution of the labeled thrombolytic protein in the patient.

Lees et al., 1989, PCT/US89101854 teach radiolabeled peptides forarterial imaging.

Sobel, 1989, PCT/US89/02656 discloses a method to locate the position ofone or more thrombi in an animal using radiolabeled, enzymaticallyinactive tissue plasminogen activator.

Stuttle, 1990, PCT/GB90/00933 discloses radioactively labeled peptidescontaining from 3 to 10 amino acids comprising the sequencearginine-glycine-aspartic acid (RGD), capable of binding to an RGDbinding site in vivo.

Maraganore et al., 1991, PCT/US90/04642 disclose a radiolabeled thrombusinhibitor comprising (a) a inhibitor moiety; (b) a linker moiety; and(c) and anion binding site moiety.

Rodwell et al., 1991, PCT/US91/03116 disclose conjugates of "molecularrecognition units" with "effector domains".

Tubis et al., 1968, Int. J. Appl. Rad. Isot. 12: 835-840 describelabeling a peptide with technetium-99m.

Sundrehagen, 1983, Int. J. Appl. Rad. Isot. 34: 1003 describes labelingpolypeptides with technetium-99m.

The use of chelating agents for radiolabeling polypeptides, and methodsfor labeling peptides and polypeptides with Tc-99m are known in theprior art and are disclosed in co-pending U.S. patent applications Ser.Nos. 07/653,012 now abandoned and 07/807,062 now U.S. Pat. No.5,443,815, which are hereby incorporated by reference.

Although optimal for radioimaging, the chemistry of Tc-99m has not beenas thoroughly studied as the chemistry of other elements and for thisreason methods of radiolabeling with technetium are not abundant. Tc-99mis normally obtained as Tc-99m pertechnetate (TcO₄ ⁻ ; technetium in the+7 oxidation state), usually from a molybdenum-99/technetium-99mgenerator. However, pertechnetate does not bind well to other compounds.Therefore, in order to radiolabel a peptide, Tc-99m pertechnetate mustbe converted to another form. Since technetium does not form a stableion in aqueous solution, it must be held in such solutions in the formof a coordination complex that has sufficient kinetic and thermodynamicstability to prevent decomposition and resulting conversion of Tc-99meither to insoluble technetium dioxide or back to pertechnetate.

Such coordination complexes of Tc-99m (in the +1 to +6 oxidation states)are known. However, many of these complexes are inappropriate forradiolabeling due to the molecular geometry of the coordination complex.For the purpose of radiolabeling, it is particularly advantageous forthe coordination complex to be formed as a chelate in which all of thedonor groups surrounding the technetium ion are provided by a singlechelating ligand. This allows the chelated Tc-99m to be covalently boundto a peptide through a single linker between the chelator and thepeptide.

These ligands are sometimes referred to as bifunctional chelating agentshaving a chelating portion and a linking portion. Such compounds areknown in the prior art.

Byrne et al., U.S. Pat. No. 4,434,151 describe homocysteinethiolactone-derived bifunctional chelating agents that can coupleradionuclides to terminal amino-containing compounds that are capable oflocalizing in an organ or tissue to be imaged.

Fritzberg, U.S. Pat. No. 4,444,690 describes a series oftechnetium-chelating agents based on 2,3-bis(mercaptoacetamido)propanoate.

Byrne et al., U.S. Pat. Nos. 4,571,430 describe novel homocysteinethiolactone bifunctional chelating agents for chelating radionuclidesthat can couple radionuclides to terminal amino-containing compoundsthat are capable of localizing in an organ or tissue to be imaged.

Byrne et al., U.S. Pat. Nos. 4,575,556 describe novel homocysteinethiolactone bifunctional chelating agents for chelating radionuclidesthat can couple radionuclides to terminal amino-containing compoundsthat are capable of localizing in an organ or tissue to be imaged.

Davison et al., U.S. Pat. No. 4,673,562 describe technetium chelatingcomplexes of bisamido-bisthiol-ligands and salts thereof, used primarilyas renal function monitoring agents.

Nicolotti et al., U.S. Pat. No. 4,861,869 describe bifunctional couplingagents useful in forming conjugates with biological molecules such asantibodies.

Fritzberg et al., U.S. Pat. No. 4,965,392 describe various S-protectedmercaptoacetylglycylglycine-based chelators for labeling proteins.

Fritzberg et al., European Patent Application No. 86100360.6 describedithiol, diamino, or diamidocarboxylic acid or amine complexes usefulfor making technetium-labeled imaging agents.

Dean et al., 1989, PCT/US89/02634 describe bifunctional coupling agentsfor radiolabeling proteins and peptides.

Flanagan et al., European Patent Application No. 90306428.5 discloseTc-99m labeling of synthetic peptide fragments via a set of organicchelating molecules.

Albert et al., European Patent Application No. WO 91/01144 discloseradioimaging using radiolabeled peptides related to growth factors,hormones, interferons and cytokines and comprised of a specificrecognition peptide covalently linked to a radionuclide chelating group.

Dean, co-pending U.S. patent application Ser. No. 07/653,012 nowabandoned teaches reagents and methods for preparing peptides comprisinga Tc-99m chelating group covalently linked to a specific binding peptidefor radioimaging in vivo, and is hereby incorporated by reference.

(It is noted that all of these procedures would be expected to formanionic complexes of technetium in the +5 oxidation state.)

Baidoo & Lever, 1990, Bioconjugate Chem. 1: 132-137 describe a methodfor labeling biomolecules using a bisamine bisthiol group that gives acationic technetium complex.

It is possible to radiolabel a peptide by simply adding athiol-containing moiety such as cysteine or mercaptoacetic acid. Suchprocedures have been described in the prior art.

Schochat et al., U.S. Pat. No. 5,061,641 disclose direct radiolabelingof proteins comprised of at least one "pendent" sulfhydryl group.

Dean et al., co-pending U.S. patent application Ser. No. 07/807,062teach radiolabeling peptides via attached groups containing free thiols,and is incorporated herein by reference.

Goedemans et al., PCT Application No. WO 89/07456 describe radiolabelingproteins using cyclic thiol compounds, particularly 2-iminothiolane andderivatives.

Thornback et al., EPC Application No. 90402206.8 describe preparationand use of radiolabeled proteins or peptides using thiol-containingcompounds, particularly 2-iminothiolane.

Stuttle, PCT Application No. WO 90/15818 describes Tc-99m labeling ofRGD-containing oligopeptides.

Again it is noted that in all of these cases the expected Tc-99m labeledspecies would be an anionic complex.

The binding of certain peptides to their target entities is sensitive tocharge modification of the peptide. Thus, it is disadvantageous in somecases to radiolabel a peptide with Tc-99m via a chelator that will forma charged Tc-99m complex. It is advantageous in certain cases to use achelator that will form an electrically neutral or uncharged Tc-99mcomplex.

This invention provides chelators for Tc-99m which may be used toprepare Tc-99m labeled peptides in which the Tc-99m is held as a neutralchelate complex.

Some chelators said to form neutral Tc-99m complexes have been describedin the prior art.

Burns et al., 1985, European Patent Application 85104959.3 describebisamine bisthiol compounds for making small neutral Tc-99m brainimaging agents.

Kung et al., 1986, European Patent Application 86105920.2 describebisamine bisthiol compounds for making small neutral Tc-99m imagingagents.

Bergstein et al., 1988, European Patent Application 88102252.9 describebisamine bisthiol compounds for making small neutral Tc-99m brainimaging agents.

Bryson et al., 1988, Inorg. Chem. 27: 2154-2161 describe neutralcomplexes of technetium-99 which are unstable to excess ligand.

Misra et al., 1989, Tet. Let. 30: 1885-1888 describe bisamine bisthiolcompounds for radiolabeling purposes.

Bryson et al., 1990, Inorg. Chem. 22: 2948-2951 describe chelatorscontaining two amide groups, a thiol group and a substituted pyridinethat may form neutral Tc-99 complexes.

Taylor et al., 1990, J. Nucl. Med. 31: 885 (Abst) describe a neutralTc-99m complex for brain imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates hypercholsterolic and normal rabbit aortae stainedwith Sudan IV.

FIG. 2 illustrates hypercholesterolic and normal rabbit aortae imaged invivo with P215 for 2.5 h

FIG. 3 illustrates hypercholesterolic and normal rabbit aortae imaged excorpora with P215.

FIG. 4 illustrates a thrombus imaged in vivo in a dog leg with Tc-99mlabeled P357.

SUMMARY OF THE INVENTION

The present invention provides scintigraphic imaging agents that areradioactively-labeled peptides. The radiolabeled peptides of theinvention are comprised of peptides that specifically bind to a targetin vivo and are covalently linked to a radiolabel-binding moiety whereinthe moiety binds a radioisotope. It is a particular advantage in thepresent invention that the complex of the radiolabel-binding moiety andthe radiolabel is electrically neutral, thereby avoiding interference ofthe covalently linked radiolabeled complex with the specific bindingproperties of the specific binding peptide.

In a first aspect of the present invention, radiolabeled peptides areprovided capable of imaging sites within a mammalian body. The peptidesare comprised of a specific binding peptide having an amino acidsequence and a radiolabel-binding moiety covalently linked to thepeptide. Further, the complex of the radiolabel-binding moiety and theradiolabel is electrically neutral. In a preferred embodiment, thepeptide is covalently linked to the radiolabel-binding moiety via anamino acid, most preferably glycine. In another preferred embodiment,the radiolabel is technetium-99m.

In a second embodiment, the invention provides a radiolabeled peptidefor imaging sites within a mammalian body, comprising a specific bindingpeptide and a radiolabel-binding moiety of formula: ##STR1## [forpurposes of this invention, radiolabel-binding moieties having thisstructure will be referred to as picolinic acid (Pic)-based moieties] or##STR2## [purposes of this invention, radiolabel-binding moieties havingthis structure will be referred to as picolylamine (Pica)-basedmoieties] wherein X is H or a protecting group; (amino acid) is anyamino acid; the radiolabel-binding moiety is covalently linked to thepeptide and the complex of the radiolabel-binding moiety and theradiolabel is electrically neutral. In a preferred embodiment, the aminoacid is glycine and X is an acetamidomethyl protecting group. Inadditional preferred embodiments, the peptide is covalently linked tothe radiolabel-binding moiety via an amino acid, most preferablyglycine, and the radiolabel is technetium-99m.

In yet another embodiment of the invention, a radiolabeled peptide isprovided for imaging sites within a mammalian body, comprising aspecific binding peptide and a bisamino bisthiol radiolabel-bindingmoiety covalently linked to the peptide. The bisamino bisthiolradiolabel-binding moiety in this embodiment of the invention has aformula selected from the group consisting of: ##STR3## wherein each Rcan be independently H, CH₃ or C₂ H₅ ; each (pgp)^(s) can beindependently a thiol protecting group or H; m, n and p areindependently 2 or 3; A is linear or cyclic lower alkyl, aryl,heterocyclyl, combinations or substituted derivatives thereof; and X ispeptide; ##STR4## wherein each R is independently H, CH₃ or C₂ H₅ ; m, nand p are independently 2 or 3; A is linear or cyclic lower alkyl, aryl,heterocyclyl, combinations or substituted derivatives thereof; V is H orCO-peptide; R' is H or peptide; provided that when V is H, R' is peptideand when R' is H, V is peptide. [For purposes of this invention,radiolabel-binding moieties having these structures will be referred toas "BAT" moieties]. In a preferred embodiment, the peptide is covalentlylinked to the radiolabel-binding moiety via an amino acid, mostpreferably glycine, and the radiolabel is technetium-99m.

In preferred embodiments of the aforementioned aspects of thisinvention, the specific binding compound is a peptide is comprised ofbetween 3 and 100 amino acids. The most preferred embodiment of theradiolabel is technetium-99m.

Specific-binding peptides provided by the invention include but are notlimited to peptides having the following sequences:

formyl-MLF

(VGVAPG)₃ amide

(VPGVG)₄ amide

RALVDTLKFVTQAEGAKamide SEQ ID NO.:1

RALVDTEFKVKQEAGAKamide SEQ ID NO.:2

PLARITLPDFRLPEIAIPamide SEQ ID NO.:3

GQQHHLGGAKAGDV SEQ ID NO.:4

PLYKKIIKKLLES SEQ ID NO.:5

LRALVDTLKamide SEQ ID NO.:6

GGGLRALVDTLKamide SEQ ID NO.:7

GGGLRALVDTLKFVTQAEGAKamide SEQ ID NO.:8

GGGRALVDTLKALVDTLamide SEQ ID NO.:9

GHRPLDKKREEAPSLRPAPPPISGGGYR SEQ ID NO.:10

PSPSPIHPAHHKRDRRQamide SEQ ID NO.:11

GGGF_(D).Cpa.YW_(D) KTFTamide SEQ ID NO.:12 ##STR5## [SYNRGDSTC]₃ -TSEAGGGLRALVDTLKamide SEQ ID NO.:14

GCGGGLRALVDTLKamide SEQ ID NO.:15

GCYRALVDTLKFVTQAEGAKamide SEQ ID NO.:16

GC(VGVAPG)₃ amide

The reagents of the invention may be formed wherein the specific bindingcompounds or the radiolabel-binding moieties are covalently linked to apolyvalent linking moiety. Polyvalent linking moieties of the inventionare comprised of at least 2 identical linker functional groups capableof covalently bonding to specific binding compounds orradiolabel-binding moieties. Preferred linker functional groups areprimary or secondary amines, hydroxyl groups, carboxylic acid groups orthiol-reactive groups. In preferred embodiments, the polyvalent linkingmoieties are comprised of bis-succinimdylmethylether (BSME),4-(2,2-dimethylacetyl)benzoic acid (DMAB), tris(succinimidylethyl)amine(TSEA) and N-[2-(N',N'-bis(2-succinimidoethyl)aminoethyl)]-N⁶,N⁹-bis(2-methyl-2-mercaptopropyl)-6,9-diazanonanamide (BAT-BS).

The invention also comprises complexes of the peptides of the inventionwith Tc-99m and methods for radiolabeling the peptides of the inventionwith Tc-99m. Radiolabeled complexes provided by the invention are formedby reacting the peptides of the invention with Tc-99m in the presence ofa reducing agent. Preferred reducing agents include but are not limitedto dithionite ion, stannous ion, and ferrous ion. Complexes of theinvention are also formed by labeling the peptides of the invention withTc-99m by ligand exchange of a prereduced Tc-99m complex as providedherein.

The invention also provides kits for preparing the peptides of theinvention radiolabeled with Tc-99m. Kits for labeling the peptide of theinvention with Tc-99m are comprised of a sealed vial containing apredetermined quantity of a peptide of the invention and a sufficientamount of reducing agent to label the peptide with Tc-99m.

This invention provides methods for preparing peptides of the inventionby chemical synthesis in vitro. In a preferred embodiment, peptides aresynthesized by solid phase peptide synthesis.

This invention provides methods for using Tc-99m labeled peptides forimaging a site within a mammalian body by obtaining in vivo gammascintigraphic images. These methods comprise administering an effectivediagnostic amount of a Tc-99m radiolabeled peptide of the invention anddetecting the gamma radiation emitted by the Tc-99m localized at thesite within the mammalian body.

Compositions of matter comprising radiolabel-binding moieties that forman electrically neutral complex with a radioisotope are also provided bythe invention. In a preferred embodiment, the radioisotope is Tc-99m.Additional preferred embodiments include bisamine bisthiol derivativesand picolinic acid and picolylamine derivatives described herein.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides Tc-99m labeled peptides for imagingtarget sites within a mammalian body comprising an amino acid sequencecovalently linked to a radiolabel-binding moiety wherein theradiolabel-binding moiety binds a radioisotope and forms an electricallyneutral complex.

Labeling with Tc-99m is an advantage of the present invention becausethe nuclear and radioactive properties of this isotope make it an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a ⁹⁹ Mo-^(99m) Tc generator. Other radionuclides known inthe prior art have effective half-lives which are much longer (forexample, ¹¹¹ In, which has a half-life of 67.4 h) or are toxic (forexample, ¹²⁵ I).

In the radiolabel binding moieties and peptides covalently linked tosuch moieties that contain a thiol covalently linked to a thiolprotecting groups [(pgp)^(s) ] provided by the invention, thethiol-protecting groups may be the same or different and may be but arenot limited to:

--CH₂ -aryl (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH-(aryl)₂, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--C-(aryl)₃, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH₂ -(4-methoxyphenyl);

--CH-(4-pyridyl)(phenyl)₂ ;

--C(CH₃)₃

-9-phenylfluorenyl;

--CH₂ NHCOR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --NHCOOR (R is unsubstituted or substituted alkyl or aryl);

--CONHR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --S--CH₂ -phenyl

Preferred protecting groups have the formula --CH₂ --NHCOR wherein R isa lower alkyl having between 1 and 8 carbon atoms, phenyl orphenyl-substituted with lower alkyl, hydroxyl, lower alkoxy, carboxy, orlower alkoxycarbonyl. The most preferred protecting group is anacetamidomethyl group.

Each specific-binding peptide-containing embodiment of the invention iscomprised of a sequence of amino acids. The term amino acid as used inthis invention is intended to include all L- and D- amino acids,naturally occurring and otherwise.

Peptides of the present invention can be chemically synthesized invitro. Peptides of the present invention can generally advantageously beprepared on an amino acid synthesizer. The peptides of this inventioncan be synthesized wherein the radiolabel-binding moiety is covalentlylinked to the peptide during chemical synthesis in vitro, usingtechniques well known to those with skill in the art. Such peptidescovalently-linked to the radiolabel-binding moiety during synthesis areadvantageous because specific sites of covalent linkage can bedetermined.

Radiolabel binding moieties of the invention may be introduced into thetarget specific peptide during peptide synthesis. For embodiments [e.g.,Pic-Gly-Cys(protecting group)-] comprising picolinic acid (Pic-), theradiolabel-binding moiety can be synthesized as the last (i.e.,amino-terminal) residue in the synthesis. In addition, the picolinicacid-containing radiolabel-binding moiety may be covalently linked tothe ε-amino group of lysine to give, for example,αN(Fmoc)-Lys-εN[Pic-Gly-Cys(protecting group)], which may beincorporated at any position in the peptide chain. This sequence isparticularly advantageous as it affords an easy mode of incorporationinto the target binding peptide.

Similarly, the picolylamine (Pica)-containing radiolabel-binding moiety[-Cys(protecting group)-Gly-Pica] can be prepared during peptidesynthesis by including the sequence [-Cys(protecting group)-Gly-] at thecarboxyl terminus of the peptide chain. Following cleavage of thepeptide from the resin the carboxyl terminus of the peptide is activatedand coupled to picolylamine. This synthetic route requires that reactiveside-chain functionalities remain masked (protected) and do not reactduring the conjugation of the picolylamine.

Examples of small synthetic peptides containing the Pic-Gly-Cys- and-Cys-Gly-Pica chelators are provided in the Examples hereinbelow. Thisinvention provides for the incorporation of these chelators intovirtually any peptide, resulting in a radiolabeled peptide having Tc-99mheld as neutral complex.

This invention also provides specific-binding small synthetic peptideswhich incorporate bisamine bisthiol (BAT) chelators which may be labeledwith Tc-99m, resulting in a radiolabeled peptide having Tc-99m held asneutral complex. Examples of small synthetic peptides containing theseBAT chelators as radiolabel-binding moiety are provided in the Exampleshereinbelow.

In forming a complex of radioactive technetium with the peptides of thisinvention, the technetium complex, preferably a salt of Tc-99mpertechnetate, is reacted with the peptides of this invention in thepresence of a reducing agent. Preferred reducing agents are dithionite,stannous and ferrous ions, the most preferred reducing agent is stannouschloride. In an additional preferred embodiment, the reducing agent is asolid-phase reducing agent. Complexes and means for preparing suchcomplexes are conveniently provided in a kit form comprising a sealedvial containing a predetermined quantity of a peptide of the inventionto be labeled and a sufficient amount of reducing agent to label thepeptide with Tc-99m. Alternatively, the complex may be formed byreacting a peptide of this invention with a pre-formed labile complex oftechnetium and another compound known as a transfer ligand. This processis known as ligand exchange and is well known to those skilled in theart. The labile complex may be formed using such transfer ligands astartrate, citrate, gluconate or mannitol, for example. Among the Tc-99mpertechnetate salts useful with the present invention are included thealkali metal salts such as the sodium salt, or ammonium salts or loweralkyl ammonium salts.

In a preferred embodiment of the invention, a kit for preparingtechnetium-labeled peptides is provided. The peptides of the inventioncan be chemically synthesized using methods and means well-known tothose with skill in the art and described hereinbelow. Peptides thusprepared are comprised of between 3 and 100 amino acid residues, and arecovalently linked to a radiolabel-binding moiety wherein theradiolabel-binding moiety binds a radioisotope. An appropriate amount ofthe peptide is introduced into a vial containing a reducing agent, suchas stannous chloride or a solid-phase reducing agent, in an amountsufficient to label the peptide with Tc-99m. An appropriate amount of atransfer ligand as described (such as tartrate, citrate, gluconate ormannitol, for example) can also be included. Technetium-labeled peptidesaccording to the present invention can be prepared by the addition of anappropriate amount of Tc-99m or Tc-99m complex into the vials andreaction under conditions described in Example 3 hereinbelow.

Radioactively labeled peptides provided by the present invention areprovided having a suitable amount of radioactivity. In forming Tc-99mradioactive complexes, it is generally preferred to form radioactivecomplexes in solutions containing radioactivity at concentrations offrom about 0.01 millicurie (mCi) to 100 mCi per mL.

Technetium-labeled peptides provided by the present invention can beused for visualizing sites in a mammalian body. In accordance with thisinvention, the technetium-labeled peptides or neutral complexes thereofare administered in a single unit injectable dose. Any of the commoncarriers known to those with skill in the art, such as sterile salinesolution or plasma, can be utilized after radiolabeling for preparingthe injectable solution to diagnostically image various organs, tumorsand the like in accordance with this invention. Generally, the unit doseto be administered has a radioactivity of about 0.01 mCi to about 100mCi, preferably 1 mCi to 20 mCi. The solution to be injected at unitdosage is from about 0.01 mL to about 10 mL After intravenousadministration, imaging of the organ or tumor in vivo can take place ina matter of a few minutes. However, imaging can take place, if desired,in hours or even longer, after the radiolabeled peptide is injected intoa patient. In most instances, a sufficient amount of the administereddose will accumulate in the area to be imaged within about 0.1 of anhour to permit the taking of scintiphotos. Any conventional method ofscintigraphic imaging for diagnostic purposes can be utilized inaccordance with this invention.

The technetium-labeled peptides and complexes provided by the inventionmay be administered intravenously in any conventional medium forintravenous injection such as an aqueous saline medium, or in bloodplasma medium. Such medium may also contain conventional pharmaceuticaladjunct materials such as, for example, pharmaceutically acceptablesalts to adjust the osmotic pressure, buffers, preservatives and thelike. Among the preferred media are normal saline and plasma.

The methods for making and labeling these compounds are more fullyillustrated in the following Examples. These Examples illustrate certainaspects of the above-described method and advantageous results. TheseExamples are shown by way of illustration and not by way of limitation.

EXAMPLE 1 Synthesis of BAT Chelators

A. Synthesis ofN-Boc-N'-(5-carboxypentyl)-N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine

a. 2-methyl-2-(triphenylmethylthio)propanal

Triphenylmethylmercaptan (362.94 g, 1.31 mol, 100 mol %) dissolved inanhydrous THF (2 L) was cooled in an ice bath under argon. Sodiumhydride (60% in oil; 54.39 g, 1.35 mol, 104 mol %) was added in portionsover 20 min. 2-bromo-2-methylpropanal (206.06 g, 1.36 mol, 104 mol %;see Stevens & Gillis, 1957, J. Amer. Chem. Soc. 79: 3448-51) was thenadded slowly over 20 min. The reaction mixture was allowed to warm toroom temperature and stirred for 12 hours. The reaction was quenchedwith water (1 L) and extracted with diethyl ether (3×1 L). The etherextracts were combined, washed with saturated NaCl solution (500 mL),dried over Na₂ SO₄ and filtered. The solvent was removed under reducedpressure to afford a thick orange oil. The crude oil was dissolved intoluene (200 mL) and diluted to 2 L with hot hexanes. The mixture wasfiltered through a sintered glass funnel and cooled at -5° C. for 12hours. The white crystalline solid which formed was removed byfiltration to afford 266.36 g (59% yield) of the title compound. Themelting point of the resulting compound was determined to be 83-85° C.Nuclear magnetic resonance characterization experiments yielded thefollowing molecular signature:

¹ H NMR(300 MHz, CDCl₃): δ1.24(s, 6H, 2CH₃), 7.2-7.35 (m, 9H), 7.59-7.62(m,6H), 8.69 (s, H, --COH)

¹³ C NMR (75 MH_(x), CDCl₃): δ22.86, 55.66, 67.48, 126.85, 127.75,129.72, 144.79, 197.31.

b. N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine.

Ethylenediamine (1.3 mL, 0.0194 mol, 100 mol %) was added to2-methyl-2-(triphenylmethylthio)propanal (13.86 g, 0.0401 mol, 206 mol%) dissolved in methanol (40 mL) and anhydrous THF (40 mL) under argon,and the pH was adjusted to pH 6 by dropwise addition of acetic acid. Thesolution was stirred for 20 min at 20° C. Sodium cyanoborohydride (1.22g, 0.0194 mol, 100 mol %) was added and the reaction was stirred at roomtemperature for 3 hours. Additional sodium cyanoborohydride (1.08 g) wasadded and the reaction was stirred at 20° C. for 17 hours. A finalportion of sodium cyanoborohydride (1.02 g) was added and the reactionheated at reflux under argon for 6 hours. The reaction was quenched with0.5 M HCl (100 mL) and extracted with ethyl acetate (2×100 mL). Theorganic extracts were combined, sequentially washed with 2 M NaOH (60mL), saturated NaCl solution (60 mL), dried (Na₂ SO₄), and filtered. Thesolvent was removed under reduced pressure to give 16.67 g of crudeproduct which was crystallized from toluene/hexanes to afford 10.20 g(73% yield) of white crystals of the title compound. The melting pointof the resulting compound was determined to be 83-86° C. FABMS analysisyielded an m/z of 721 (MH+). Nuclear magnetic resonance characterizationexperiments yielded the following molecular signature:

¹ H NMR (300 MH_(z), CDCl₃): δ1.12 (s, 12H, 4 CH₃), 1.64 (s, 4H, N--CH₂--C(Me)₂ --S), 2.52 (s, 4H, N--CH₂ --CH₂ --N), 5.31 (S, 2H, 2-NH),7.12-7.30 (m, 18H, Ar), 7.62-7.65 (m, 12H, Ar).

c.N-(5-carboethoxypentyl)-N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine

K₂ CO₃ (1.92 g, 13.9 mmol, 100 mol %) was added toN,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine (10.03 g,13.9 mmol) in CH₃ CN (60 mL), followed by ethyl 5-bromovalerate (3.30mL, 20.8 mmol, 150 mol %). The reaction was heated at reflux under argonovernight. The solution was then concentrated to a paste and partitionedbetween 0.25 M KOH (100 mL) and ethyl acetate (100 mL). The aqueouslayer was extracted with ethyl acetate (1×50 mL) and the combined ethylacetate layers were washed with 50 mL water and NaCl solution (2×50 mL),dried with Na₂ SO₄ and concentrated to an orange oil. Purification byflash chromatography (300 g flash silica, 100% CHCl₃ to 5% MeOH/CHCl₃)gave pure title compound (7.75 g, 66% yield). FABMS analysis yielded an(MH+) of 849 (compared with a calculated molecular weight of 849.24 forthe compound C₅₅ H₆₄ N₂ O₂ S₂).

d.N-Boc-N'-(5-carboxypentyl)-N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine

1M KOH (25 mL, 25.0 mmol, 274 mol %) was added toN-(5-carboethoxypentyl)-N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine (7.75 g, 9.13 mmol) in dioxane (200 mL), followed bywater (250 mL). Dioxane was then added dropwise with stirring until ahomogeneous solution was obtained. The reaction was heated at a slowreflux overnight. Most of the dioxane was removed by rotary evaporationand the pH of solution was adjusted to ˜7-8 with 1 M KH₂ PO₄ andsaturated NaHCO₃. The solution was then extracted with ethyl acetate(3×75 mL) and the combined organic layers were washed with NaCl solution(50 mL), dried with Na₂ SO₄ and concentrated to a foam/solid (6.35 g,85% yield).

To the crude product from the above reaction was added (BOC)₂ O (3.35 g,15.4 mmol, 200 mol %), CH₃ CN (50 mL) and methylene chloride (50 mL),followed by triethylamine (1.0 mL, 7.2 mmol, 93 mol %). The reaction wasstirred at room temperature under argon overnight. The reaction solutionwas then concentrated and partitioned between water (100 mL) and ethylacetate (50 mL). The aqueous layer was extracted with ethyl acetate(1×50 mL) and the combined ethyl acetate layers were .washed with 5%citric acid and NaCl solution (50 mL each), then dried (Na₂ SO₄) andconcentrated to an orange oil. Purification by flash chromatography (200g flash silica, 100% CDCl₃ to 5% methanol/chloroform) gave pure titlecompound (2.58 g, 36% yield). FABMS analysis gave an (MH+) of 921(compared with the calculated value of 921.31 for the compound C₅₈ H₆₈N₂ O₄ S₂).

B. Synthesis ofN-Boc-N'-(5-carboxypentyl)-N,N'-bis-[2-(4-methoxybenzylthio)-2-methylpropyl]ethylenediamine

a. N,N'-bis-[2-(4-methoxybenzylthio)-2-methylpropyl]-ethylenediamine

A solution of N,N'-bis(2-mercapto-2-methylpropyl)ethylenediamine (11.23g, 47.5 mmol; see, DiZio et al., 1991, Bioconjugate Chem 2: 353 andCorbin et al., 1976, J. Org. Chem. 41: 489) in methanol (500 mL) wascooled in ice/water bath and then saturated with gaseous ammonia over 45min. To this was added 4-methoxybenzyl chloride (17.0 mL, 125 mmol, 264mol %). The reaction was allowed to warm to room temperature overnightwith stirring under argon. The solution was concentrated to a paste andthen partitioned between diethyl ether (150 mL) and 0.5 M KOH (200 mL).The aqueous layer was further extracted with diethyl ether (2×50 mL).The combined organic layers were washed with NaCl solution andconcentrated to a clear colorless oil. The oil dissolved in diethylether (200 mL) and then acidified with 4.0 M HCl in dioxane until nofurther precipitation was seen. The white precipitate was collected byfiltration and washed with diethyl ether. The white solid wasrecrystallized from hot water at a pH of -2. The product was collectedby filtration to afford 29.94 g as a mix of mono- and di- HCl salts. TheHCl salts were partitioned between 1 M KOH (100 mL) and ethyl acetate(100 mL). The aqueous was extracted with ethyl acetate (2×30 mL) and thecombined organic layers were washed with NaCl solution, dried with Na₂SO₄ and concentrated to give pure product as the free base as a lightyellow oil (18.53 g, 82% yield). Nuclear magnetic resonancecharacterization experiments yielded the following molecular signature:

¹ H NMR (300 MHz, CDCL₃): δ7.25 (d, 4H, J=9), 6.83 (d, 4H, J=9), 3.78(s, 6H), 3.67 (s, 4H), 2.63 (s, 4H), 2.56 (s, 4H), 1.34 (s, 12H).

b.N-(5-carboethoxypentyl)-N,N'-bis-[2-(4-methoxybenzylthio)-2-methylpropyl]ethylenediamine

To N,N'-bis-[2-(4-methoxybenzylthio)-2-methylpropyl]-ethylenediamine(4.13 g, 8.66 mmol) in CH₃ CN (50 mL) was added K₂ CO₃ (1.21 g, 8.75mmol, 101 mol %) followed by ethyl 5-bromovalerate (2.80 mL, 17.7 mmol,204 mol %). The reaction was stirred at reflux overnight and was thenconcentrated to a paste in vacuo. The residue was partitioned betweenethyl acetate (100 mL) and 0.5 M KOH (100 mL). The aqueous layer wasextracted with ethyl acetate (1×50 mL) and the combined organic layerswere washed with NaCl solution (50 mL), dried with Na₂ SO₄ andconcentrated to a yellow oil (˜6 g). Purification by normal-phasepreparative HPLC (100% CHCl₃ to 5% methanol/chloroform over 25 min.)afforded pure title compound (1.759 g, 34% yield). FABMS analysis gavean (MH+) of 605 (compared with the value of 604.90 calculated for C₃₃H₅₂ N₂ O₄ S₂). Nuclear magnetic resonance characterization experimentsyielded the following molecular signature:

¹ H NMR (300 mH_(z), CDCl₃): δ7.25 (d, 4H, J=8.5), 6.83 (d, 4H, J=8.5),4.13 (q, 2H, J=7), 3.793 (s, 3H), 3.789 (s. 3H), 3.74 (s, 2H), 3.67 (s.2H), 2.6 (m, 10H), 2.31 (t, 2H, J=7), 1.6 (m, 2H), 1.5 (m 2H), 1.34 (s12H), 1.28 (t, 3H, J=37).

c.N-Boc-N'-(5-carboxypentyl)-N,N'-bis-[2-(4-methoxybenzylthio)-2-methylpropyl]ethylenediamine

ToN-(5-carboethoxypentyl)-N,N'-bis-[2-(4-methoxybenzylthio)-2-methylpropyl]ethylenediamine(586 mg, 0.969 mmol) in THF (40 mL) was added water (30 mL) and 1 M KOH(2.5 mL, 2.5 mmol, 260 mol %). The homogeneous solution was heated to aslow reflux overnight. The solution was then cooled to room temperatureand the THF was removed under rotary evaporation. The residue wasdiluted to 50 mL with H₂ O and the pH was adjusted to ˜2-3 with 1 M HCl.The solution was extracted with ethyl acetate (3×30 mL) and the combinedorganic layers were washed with NaCl solution (50 mL), dried with Na₂SO₄ and concentrated to give crude acid (422 mg, 75% yield).

To the crude product from the above reaction was added CH₃ CN (40 mL)and (BOC)₂ O (240 mg, 1.10 mmol, 150 mol %) followed by triethylamine(0.200 mL, 1.43 mmol, 196 mol %). The homogenous solution was stirred atroom temperature overnight under argon. The solution was thenconcentrated to a paste and partitioned between ethyl acetate (25 mL)and 1 M KH₂ PO₄ (25 mL). The organic layer was washed with 5% citricacid (2×25 mL) and NaCl solution (25 mL), dried with Na₂ SO₄ andconcentrated to a yellow oil. Purification by flash chromatography (50mL flash silica gel, 100% chloroform to 15% methanol/chloroform) gavepure title compound (344 mg, 70% yield). FABMS analysis gave an (MH+) of677 (compared to the value of 676.97 calculated for the compound C₃₆ H₅₆N₂ O₆ S₂). Nuclear magnetic resonance characterization experimentsyielded the following molecular signature:

¹ H NMR (300 MHz, CDCl₃): δ7.20 (d, 4H, J=7), 6.79 (d, 4H, J=7), 3.75(S, 3H), 3.74 (S, 3H), 3.68 (M, 4H), 3.35 (M, 4H), 2.65 (M, 2H), 2.53(M, 4H), 2.31 (M, 2H), 1.59 (M, 2H), 1.43 (S, 11H), 1.30 (S, 6H), 1.26(S, 6H)

C. Synthesis of BAT-BM(N-[2-(N',N'-bis(2-maleimidoethyl)aminoethyl)]-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanamide)

BAT-BM was prepared as follows. BAT acid (N⁹ -(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanoic acid)(10.03 g, 10.89 mmol) and 75 mL of dry methylene chloride (CH₂ Cl₂) wereadded to a 250 mL round-bottomed flask equipped with magnetic stir barand argon balloon. To this solution was added diisopropylcarbodiimide(3.40 mL, 21.7 mmol, 199 mole %), followed by N-hydroxy-succinimide(3.12 g, 27.1 mmol. 249 mole %). This solution was observed to becomecloudy within 1 h, and was further incubated with stirring for a totalof 4 h at room temperature. A solution of tris(2-aminoethyl)amine (30mL, 200 mmol, 1840 mole %) in 30 mL methylene chloride was then addedand stirring continued overnight. The reaction mixture was thenconcentrated under reduced pressure, and the residue partitioned betweenethylacetate (150 mL) and 0.5M potassium carbonate (K₂ CO₃ ; 150 mL).The organic layer was separated, washed with brine and concentrated togive the crude productN-[2-(N',N'-bis(2-aminoethyl)aminoethyl)]-N'-(t-butoxycarbonyl)-N⁶,N.sup.9-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanamide as afoam/oil.

This crude product was added to a 1000 mL round-bottomed flask, equippedwith magnetic stir bar, containing 300 mL THF, and then 30 mL saturatedsodium bicarbonate (NaHCO₃), 100 mL water and N-methoxycarbonylmaleimide(6.13 g, 39.5 mmol, 363 mole %) were added. This heterogeneous mixturewas stirred overnight at room temperature. THF was removed from themixture by rotary evaporation, and the aqueous residue was twiceextracted with ethylacetate (2×75 mL). The combined organic layers ofthese extractions were washed with brine, dried over sodium sulfate,filtered through a medium frit and concentrated to about 12 g of crudeproduct. Purification by liquid chromatography (250 g silicondioxide/eluted with a gradient of chloroform→2% methanol in chloroform)afforded 5.3 g of pure product(N-[2-(N',N'-bis(2-maleimidoethyl)aminoethyl)]-N⁹-(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanamide(equivalent to 40% yield), along with approximately 5 g of crude productthat can be repurified to yield pure product. Chemical analysis of thepurified product confirmed its identity as BAT-BM as follows:

¹ H NMR (200 mHz, CDCl₃): δ0.91 (12H,s), 1.38 (9H,s), 1.2-1.6 (4H,m),2.06 (2H,s), 2.18 (2H,t,J=7), 2.31 (4H,m), 2.55 (2H,t,J=5), 2.61(4H,t,J=6), 2.99 (2H,s), 3.0-3.3 (4H,m), 3.46 (4H,t,J=6), 6.49(--NH,t,J=4), 6.64 (4H,s), 7.1-7.3 (18H,m), 7.6 (12H,t,J=17).

D. Synthesis or [BAT]-conjugated(εN) Lys(αN-Fmoc) [N-ε-(N⁹-t-butoxycarbonyl)-N⁶,N⁹-bis[2-methyl-2-(triphenylmethylthio)propyl]-6,9-diazanonanoyl)-N-α-Fmoc-lysine

A 100 mL single-necked round-bottomed flask, equipped with stir bar andargon balloon, was charged with N⁹ -(t-butoxycarbonyl)-N⁶,N⁹-bis[2-methyl-2-(triphenylmethylthio)propyl]-6,9-diazanonanoic acid (BATacid; 3.29 g, 3.57 mmol) in 50 mL CH₂ Cl₂ at room temperature. To thiswas added diisopropylcarbodiimide (DIC; 580 μL, 3.70 mmol, 104 mole %)followed immediately by N-hydroxysuccinimide (HOSu; 432 mg, 3.75 mmol,105 mole %). The reaction was stirred overnight at room temperatureduring which time a white precipitate developed. The mixture wasfiltered and the filtrate concentrated to a solid foam. The crude foam,in a 100 mL round-bottomed flask, was dissolved in 75 mL of a 2:1mixture of dimethoxyethane and water. To this homogeneous solution wasadded N-α-Fmoc-lysine hydrochloride (1.52 g, 3.75 mmol, 105 mole %)followed by K₂ CO₃ (517 mg, 3.74 mmol, 105 mole %), and the yellowsolution stirred overnight at room temperature. The solution was thenpoured into a 250 mL erlenmeyer flask containing 100 mL of ethyl acetateand 100 mL of water. The organic layer was separated and the aqueouslayer further extracted with 50 mL ethyl acetate. The combined organiclayers were washed once with brine (100 mL), dried over Na₂ SO₄ andconcentrated to a yellow solid. This crude product was purified bylow-pressure liquid chromatography (150 g SiO₂, eluted with CHCl₃ -->10%methanol in CHCl₃). In this way, 3.12 g of the named compound wasprepared (69% yield). Chemical analysis of the purified productconfirmed its identity as follows:

¹ H NMR (300 MHz, CDCl₃): δ0.88 (12H,s,broad), 1.05-1.45 (19H,m),1.8-2.1 (4H,m), 1.8-2.47 (4H,m), 2.75-3.2 (6H,m), 3.94.3 (4H,m,), 7.2(22H,m), 7.6 (16H,s,bound). FABMS MH⁺ was predicted to be 1270.6 andfound to be 1272.

E. Synthesis of BAM (N¹ -(t-butoxycarbonyl)-N¹,N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-1,4,10-triazadecane

A 250 mL single-necked round-bottomed flask, equipped with a stir bar,reflux condenser and argon balloon, was charged with N¹,N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-ethylenediamine (BAT-I;10.0 g, 14.01 mmol) in 50 mL of CH₃ CN and 30 mL dioxane. To this wasadded N-(5-bromopentyl)phthalimide (8.04 g, 27.1 mmol, 194 mole %)followed by K₂ CO₃ (2.95 g, 21.3 mmol, 152 mole %). The mixture washeated at reflux under argon for two days. The reaction mixture was thenconcentrated and the residue partitioned between 150 mL water and 150 mLethyl acetate. The organic layer was separated and the aqueous layer (atpH of about 10) was further extracted with 50 mL ethyl acetate. Thecombined organic layers were washed once with brine (75 mL), dried overNa₂ CO₃ and concentrated to an oil. Purification by low-pressure liquidchromatography (300 g SiO₂, CHCl₃ -->2% methanol in CHCl₃) afforded 9.20g of 9-phthalimido-N¹,N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-1,4-diazanonane as a yellowfoam (70% yield). Chemical analysis of the purified product of thisintermediate confirmed its identity as follows:

¹ H NMR (300 MHz, CDCl₃): δ1.01 (6H,s), 1.03 (6H,s), 1.15-1.4 (2H,t),1.98 (2H,s), 2.10 (2H,s), 2.28 (2H,m), 2.45 (3H,m), 3.68 (2H,t),7.15-7.35 (18H, m), 7.62 (12H, 0), 7.72 (2H, m), 7.85 (2H,m). FABMS MH⁺was predicted to be 935.4 and found to be 936.

A 500 mL single-necked round-bottomed flask, equipped with stir bar, wascharged with 9-phthalimido-N¹,N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-1,4-diazanonane (8.83 g,9.43 mmol) in 75 mL of CH₃ CN and 20 mL CH₂ Cl₂. To this was added K₂CO₃ (1.30 g, 9.41 mmol, 100 mole %), followed by di-tert-butyldicarbonate (2.15 g, 9.85 mmol, 104 mole %), and the reaction stirred atroom temperature overnight. The reaction mixture was then concentratedand partitioned between 100 mL each of water and ethyl acetate. Theorganic layer was separated and the aqueous layer was further extractedwith 50 mL ethyl acetate. The combined organic layers were washed oncewith brine (75 mL), dried over Na₂ SO₄ and concentrated to give 9.69 gof crude 9-phthalimido-N¹ -(t-butoxycarbonyl)-N¹,N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-1,4-diazanonane as a yellowfoam (99% crude yield). This crude product was used without furtherpurification.

A 250 mL single-necked round-bottomed flask, equipped with stir bar andreflux condenser, was charged with 9-phthalimido-N¹-(t-butoxycarbonyl)-N¹,N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-1,4-diazanonane (5.50 g,5.319 mmol) in 25 mL of THF. To this was added 100 mL ethanol and 5 mLwater. The addition of water caused the starting material to precipitateout of solution. Hydrazine hydrate (1.2 mL, 24.7 mmol, 466 mole %) wasadded, and the reaction heated at reflux for two days. The reactionmixture was concentrated and partitioned between 100 mL each of waterand 0.25M K₂ CO₃. The organic layer was separated and washed once withbrine (75 mL), dried over Na₂ SO₄ and concentrated to a solid foam.Purification of the crude product by low-pressure liquid chromatography(100 g SiO₂, CHCl₃ -->5% methanol in CHCl₃, the column pre-treated with200 mL 2% triethylamine in CHCl₃) afforded 3.27 g of pure N¹-(t-butoxycarbonyl)-N¹,N⁴-bis(2-methyl-2-(triphenylmethylthio)propyl]-1,4,10-triazadecane as ayellow foam (68% yield). Chemical analysis of the purified productconfirmed its identity as follows:

¹ H NMR (300 MHz, CDCl₃): δ0.9 (12H,s), 1.2 (6H,s), 1.36 (9H,s), 2.05(4H,m), 2.24 (2H,t), 2.31 (2H,t), 2.62 (3H,t), 3.0 (2H,s,broad), 3.1(2H,s,broad), 7.2 (18H,m), 7.6 (12H,t). FABMS MH⁺ was predicted to be905.5 and found to be 906.5.

EXAMPLE 2 Solid Phase Peptide Synthesis

Solid phase peptide synthesis (SPPS) was carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with dicyclohexylcarbodiimide/hydroxybenzotriazole or2-(1H-benzo-triazol--1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and usingp-hydroxymethylphenoxymethylpolystyrene (HMP) resin forcarboxyl-terminus acids or Rink amide resin for carboxyl-terminusamides. Resin-bound products were routinely cleaved using a solutioncomprised of trifluoroacetic acid, water, thioanisole, ethanedithiol,and triethylsilane, prepared in ratios of 100:5:5:2.5:2 for 1.5-3 h atroom temperature.

Where appropriate αN-formyl groups were introduced by treating thecleaved, deprotected peptide with excess acetic anhydride in 98% formicacid and stirring for about 18 hours followed by HPLC purification.Where appropriate N-terminal acetyl groups were introduced by treatingthe free N-terminal amino peptide bound to the resin with 20% v/v aceticanhydride in NMP (N-methylpyrrolidinone) for 30 min. Where appropriate,2-chloroacetyl and 2-bromoacetyl groups were introduced either by usingthe appropriate 2-halo-acetic acid as the last residue to be coupledduring SPPS or by treating the N-terminus free amino peptide bound tothe resin with either the 2-haloaceticacid/diisopropylcarbodiimide/N-hydroxysuccinimide in NMP of the2-halo-acetic anhydride/diisopropylethylamine in NMP. Where appropriate,HPLC-purified 2-haloacetylated peptides were cyclized by stirring an0.1-1.0 mg/mL solution in bicarbonate or ammonia buffer (pH 8) with orwithout 0.5-1.0 mM EDTA for 1-48 hours, followed by acidification withacetic acid, lyophilization and HPLC purification. Where appropriate,Cys-Cys disulfide bond cyclizations were performed by treating theprecursor cysteine-free thiol peptides at 0.1 mg/mL in pH 7 buffer withaliquots of 0.006 M K₃ Fe(CN)₆ until a stable yellow color persisted.The excess oxidant was reduced with excess cysteine, the mixture waslyophilized and then purified by HPLC.

Where appropriate the "Pica" group was introduced by conjugatingpicolylamine to a precursor peptide using diisopropylcarbodiimide andN-hydroxysuccinimide. Where appropriate BAT ligands were introducedeither by using the appropriate BAT acid as the last residue to becoupled during SPPS or by treating the N-terminus free amino peptidebound to the resin with BATacid/diisopropylcarbodiimide/N-hydroxysuccinimide in NMP. Whereappropriate, [BAM] was conjugated to the peptide by first activating thepeptide carboxylate with a mixture ofdiisopropylcarbodiimide/N-hydroxysuccinimide or HBTU/HOBt in DMF, NMP orCH₂ Cl₂, followed by coupling in the presence of diisopropylethylamine,after coupling, the conjugate was deprotected as described above.

Where appropriate, BSME adducts were prepared by reacting singlethiol-containing peptides (5 to 50 mg/mL in 50 mM sodium phosphatebuffer, pH 8) with 0.5 molar equivalents of BMME(bis-maleimidomethylether) predissolved in acetonitrile at roomtemperature for approximately 1-18 hours. The solution was concentratedand the product was purified by HPLC.

Where appropriate, TSEA adducts were prepared by reacting singlethiol-containing peptide (at concentrations of 10 to 100 mg/mL peptidein DMF, or 5 to 50 mg/mL peptide in 50 mM sodium phosphate (pH8)/acetonitrile or THF) with 0.33 molar equivalents of TMEA(tris(2-maleimidoethyl)amine; Example 2) pre-dissolved in acetonitrileor DMF, with or without 1 molar equivalent of triethanolamine, at roomtemperature for approximately 1-18 h. Such reaction mixtures containingadducts were concentrated and the adducts were then purified using HPLC.

Where appropriate, BAT-BS adducts were prepared by reacting singlethiol-containing peptide (at concentrations of 2 to 50 mg/mL peptide in50 mM sodium phosphate (pH 8)/acetonitrile or THF) with 0.5 molarequivalents of BAT-BM (N-[2-(N',N'-bis(2-maleimidoethyl)aminoethyl)]-N⁹-(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanamide;Example 1) pre-dissolved in acetonitrile or THF, at room temperature forapproximately 1-18 h. The solution was then evaporated to dryness and[BAT-BS]-peptide conjugates deprotected by treatment with 10 mL TFA and0.2 mL triethylsilane for 1 h. The solution was concentrated, theproduct adducts precipitated with ether, and then purified by HPLC.

Crude peptides were purified by preparative high pressure liquidchromatography (HPLC) using a Waters Delta Pak C18 column and gradientelution using 0.1% trifluoroacetic acid (TFA) in water modified withacetonitrile. Acetonitrile was evaporated from the eluted fractionswhich were then lyophilized. The identity of each product was confirmedby fast atom bombardment mass spectroscopy (FABMS).

EXAMPLE 3 A General Method for Radiolabeling with Tc-99m

0.1 mg of a peptide prepared as in Example 2 was dissolved in 0.1 mL ofwater or 50 mM potassium phosphate buffer (pH=5, 6 or 7.4). Tc-99mgluceptate was prepared by reconstituting a Glucoscan vial (E. I. DuPontde Nemours, Inc.) with 1.0 mL of Tc-99m sodium pertechnetate containingup to 200 mCi and allowed to stand for 15 minutes at room temperature.25 μl of Tc-99m gluceptate was then added to the peptide and thereaction allowed to proceed at room temperature or at 100° C. for 15-30min and then filtered through a 0.2 μm filter.

The Tc-99m labeled peptide purity was determined by HPLC using thefollowing conditions: a Waters DeltaPure RP-18, 5μ, 150 mm×3.9 mmanalytical column was loaded with each radiolabeled peptide and thepeptides eluted at a solvent flow rate equal to 1 mL/min. Gradientelution was performed beginning with 10% solvent A (0.1% CF3COOH/H₂ O)to 40% solvent B₉₀ (0.1% CF₃ COOH/90% CH₃ CN/H₂ O) over the course of 20min.

Radioactive components were detected by an in-line radiometric detectorlinked to an integrating recorder. Tc-99m gluceptate and Tc-99m sodiumpertechnetate elute between 1 and 4 minutes under these conditions,whereas the Tc-99m labeled peptide eluted after a much greater amount oftime.

The following Table illustrates successful Tc-99m labeling of peptidesprepared according to Example 2 using the method described herein.

    __________________________________________________________________________                               FABMS                                                                             Radiochemical                                                                        HPLC                                    Peptides                   MH.sup.+                                                                          Yield(%)*                                                                            R(min)**                                __________________________________________________________________________    formyl-MLFC.sub.Acm G.Pica  760                                                                              100    10.9, 11.5, 12.2                        Pic.GC.sub.Acm (VGVAPG).sub.3 amide                                                                      1795                                                                              100    12.4                                    Pic.GC.sub.Acm (VPGVG).sub.4 amide                                                                       1992                                                                              100    12.0                                    Pic.GC.sub.Acm RALVDTLKFVTQAEGAKamide                                                                    2183                                                                              95     17.2                                    Pic.GC.sub.Acm RALVDTEFKVKQEAGAKamide                                                                    2226                                                                              96     15.5                                    Pic.GC.sub.Acm PLARITLPDFRLPEIAIPamide                                                                    236                                                                              92     19.2                                    Pic.GC.sub.Acm GQQHHLGGAKAGDV                                                                            1838                                                                              48     12.8-16.6                               Pic.GC.sub.Acm PLYKKIIKKLLES                                                                             1910                                                                              81     10.7-14.5                               Pic.GC.sub.Acm LRALVDTLKamide                                                                            1363                                                                              92     13.0-14.5                               Pic.GC.sub.Acm GGGLRALVDTLKamide                                                                         1535                                                                              100    15.6                                    Pic.GC.sub.Acm GGGLRALVDTLKFVTQAEGAKamide                                                                2354                                                                              92     15.1                                    Pic.GC.sub.Acm GGGRALVDTLKALVDTLamide                                                                    2035                                                                              86     14.5                                    Pic.GC.sub.Acm GHRPLDKKREEAPSLRPAPPPISGGGYR                                                              3377                                                                              94     11.3                                    Pic.GC.sub.Acm PSPSPIHPAHHKRDRRQamide                                                                    2351                                                                              94     11.2, 14.4                              Pic.GC.sub.Acm GGGF.sub.D.Cpa.YW.sub.D KTFTamide                                                         1681                                                                              98     13.8-16.8                               Pic.GC.sub.Acm GGCNP.Apc.GDC                                                                             1217                                                                              69      6.6-13.7                               (Pic.SC.sub.Acm SYNRGDSTC).sub.3 -TSEA                                                                   4488                                                                              99     10.4, 11.2                              Pic.GC.sub.Muh GGGLRALVDTLKamide                                                                         1471                                                                              100    11.9                                    Pic.GCGGGLRALVDTLKamide    1350                                                                              100    11.2, 11.6                              Pic.GCYRALVDTLKFVTQAEGAKamide                                                                            2275                                                                              95     18.6, 19.1                              Pic.GC(VGVAPG).sub.3 amide 1724                                                                              95     17.3                                    [BAT]GGPLYKKIIKKLLES       2006                                                                              94      9.5                                    [BAT].Hly.GDP.HlY.GDF.amide                                                                              1209                                                                              99     10.8                                    [BAT]GHRPLDKKREEAPSLRPAPPPISGGGYR.amide                                                                  3357                                                                              93     10.4, 11.6                              [BAT]PKLEELKEKLKELLEKLKEKLA                                                                              2969                                                                              90     12.3                                    [BAT]G.Apc.GDV.Apc.GDFK.amide                                                                            1432                                                                              97     17.5                                    [BAT]PLARITLPDFRLPEIAIP.amide                                                                            2350                                                                              N.D    N.D                                     [BAT]RALVDTEFKVKQEAGAK.amide                                                                             2208                                                                              96     12.1                                    [BAT]YRALVDTLKFVTQAEGAK.amide                                                                            2329                                                                              96     13.3                                    [BAT]VPGVGVPGVGVPGVGVPGVG.amide                                                                          1974                                                                              96     11.9, 12.8                              [BAT]RALVDTLKFVTQAEGAKamide                                                                              2165                                                                              98     19.0                                    formyl-MLFK[BAT]amide       884                                                                              99     12.6                                    formyl.Thp.LF[BAM]          775                                                                              99     13.3, 13.6                              (CH.sub.3 --N)-FYW.sub.D KVE[BAM]                                                                        1171                                                                              98     12.3, 13.6                              formyl-MLFK[BAT]            884                                                                              96     11.9, 12.8                              formyl-MLFK[BAT]KKKKK.amide                                                                              1524                                                                              96     11.7, 12.2                              formyl-MLFK[BAT]GSGSGS.amide                                                                             1315                                                                              97     11.9, 12.8                              formyl-MLFK[BAT]EGE        1013                                                                              99     12.3                                    formyl-M.Dpg.F[BAM]         770                                                                              98     13.7                                    formyl-MLFK[BAT]E          1200                                                                              98     13.7                                    (formyl-MLFK[BAT]GGC.sub.Acm GGC.amide).sub.2 -BSME                                                      3477                                                                              99     11.9, 12.4                              [BAT]RALVDTLKKLKKKL.amide  2135                                                                              97     11.9                                    (CH.sub.3 CO-Y.sub.D.Apc.GDCGGC.sub.Acm GC.sub.Acm GGC.amide).sub.2           -[BAT-BS]                  3409                                                                              98     10.3                                    [BAT](VGVAPG).sub.3.amide  1778                                                                              98     10.3                                    YRALVDTLKFVTQAEGAK[BAT].amide                                                                            2329                                                                              98     11.4                                    K[BAT]D.Nal.C.sub.Me YW.sub.D KCV.sub.Me T.amide                                                         1573                                                                              97     12.0, 12.5                              [DTPA](D-Nal.SYW.sub.D KVTK[BAT]).sub.2.amide                                                            3210                                                                              97     12.1, 12.5                              [DTPA](D-Nal.SYW.sub.D KVTK[BAT]).amide                                                                  1801                                                                              96     11.8, 12.0                              [DTPA]K[BAT]D-Nal.C.sub.Me YW.sub.D KVC.sub.Me T.amide                                                   1949                                                                              96     11.8, 12.0                              [BAT-BS](ma)GGGRALVDTLKFVTQAEGAK.amide                                                                   4808                                                                              96     12.0                                    [BAT]KKLLKKLYKKIIKKLLES    2533                                                                              99     bound                                   pGlu.GVNDNEEGFFSARK[BAT].amide                                                                           1997                                                                              N.D    N.D.                                    __________________________________________________________________________    *The following labeling conditions were used with the appropriate             peptides:                                                                     1. The peptide is dissolved in 50 mM potassium phosphate buffer (pH 7.4)      and labeled at room temperature.                                              2. The peptide is dissolved in 50 mM potassium phosphate buffer (pH 7.4)      and labeled at 100° C.                                                 3. The peptide is dissolved in water and labeled at room temperature.         4. The peptide is dissolved in water and labeled at 100° C.            5. The peptide is dissolved in 50 mM potassium phosphate buffer (pH 6.0)      and labeled at 100° C.                                                 6. The peptide is dissolved in 50 mM potassium phosphate buffer (pH 5.0)      and labeled at room temperature.                                              **HPLC methods:                                                               general:                                                                          solvent A = 0.1% CF.sub.3 COOH/H.sub.2 O                                      solvent B.sub.70 = 0.1% CF.sub.3 COOH/70% CH.sub.3 CN/H.sub.2 O               solvent B.sub.90 = 0.1% CF.sub.3 COOH/90% CH.sub.3 CN/H.sub.2 O               solvent flow rate = 1 mL/min                                              Vydak column = Vydak 218TP54 RP-18, 5μ × 220 mm × 4.6 mm       analytical column with guard column                                           Brownlee column = Brownlee Spheri-5 RP-18, 5μ × 220 mm ×       4.6 mm column                                                                 Waters column = Waters Delta-Pak C18, 5μm, 39 × 150 mm               Method 1:                                                                          Brownlee column                                                                       100% A to 100% B.sub.70 in 10 min                                Method 2:                                                                          Vydak column                                                                          100% A to 100% B.sub.90 in 10 min                                Method 3:                                                                          Vydak column                                                                          100% A to 100% B.sub.70 in 10 min                                Method 4:                                                                          Brownlee column                                                                       100% A to 100% B.sub.90 in 10 min                                Method 5:                                                                          Waters column                                                                         100% A to 100% B.sub.90 in 10 min                                Single-letter abbreviations for amino acids can be found in G. Zubay,         Biochemistry (2d. ed.), 1988 (MacMillen                                       Publishing: New York) p. 33; Ac = acetyl; Pic = picolinoyl                    (pyridine-2-carbonyl) = 6-aminocaproic acid;                                  Hly = homolysine; Acm = acetamidomethyl; pGlu = pyro-glutamic acid; Mob =     4-methoxybenzyl; Pica =                                                       picolylamine (2-(aminomethyl)pyridine); Apc = L-[S-(3-aminopropyl)cysteine    ; F.sub.D = D-phenylalanine; W.sub.D =                                        D-tryptophan; Y.sub.D = D-tyrosine; Cpa = L-(4-chlorophenyl)alanine; Thp      = 4-amino-tetrahydrothiopyran-4-                                              carboxylic acid; ma = mercaptoacetic acid; D-Nal = D-2-naphthylalanine;       Dpg = dipropylglycine; Nle =                                                  norleucine; BAT = N.sup.6,N.sup.9 -bis(2-mercapto-2-methylpropyl)-6,9-diaz    anonanoic acid; BAT acid (protected) = N.sup.9 -                              (t-butoxycarbonyl)-N.sup.6,N.sup.9 -bis(2-methyl-2-triphenylmethylthioprop    yl)-6,9-diazanonanoic acid; BAM = N.sup.1,N.sup.4 -                           bis(2-mercapto-2-methylpropyl)-1,4,10-triazadecane; BAM (protected) =         N.sup.1 -(t-butoxycarbonyl)-N.sup.1,N.sup.4 -                                 bis(2-methyl-2-triphenylmethylthiopropyl)-1,4,10-triazadecane; [BAT-BM] =     N-[2-(N',N'-                                                                  bis(2-maleimidoethyl)aminoethyl]-N.sup.9 -(t-butoxycarbonyl)-N.sup.6,N.sup    .9 -bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-                            diazanonanamide; [BAT-BS] = N-[2-(N',N'-bis(2-succinimidoethyl)aminoethyl]    -N.sup.6,N.sup.9 -(2-mercapto-2-                                              methylpropyl)-6,9-diazanonanamide; [BMME] = bis-maleimidomethylether;         [BSME] = bis-                                                                 succinimidomethylether; [DTPA] = diethylenetriaminepentaacetic acid       

EXAMPLE 4 Localization and In Vivo Imaging of Atherosclerotic Plaqueusing Tc-99m Labeled Compound P215 in the Hypercholesterol Rabbit Model

Twenty-two New Zealand White (NZW) rabbits of both sexes and weighing2-3 kg were divided into two groups. The control group consisted of 6rabbits that were housed and fed commercial rabbit chow (Purina).Sixteen rabbits, the HC group, were fed a standardized, cholesterol-richdiet (rabbit chow mixed to a 1% w/w concentration of cholesterol) fromseven weeks until 28 weeks of age. All animals were given water adlibitum.

Tc-99m labeled P215 ([BAT]RALVDTLKFVTQAEGAK.amide) was prepared asdescribed above. Approximately 250-400 μg of peptide was labeled with140-160 mCi of Tc-99m and prepared in unit doses of 7-8 mCi (12.5-20.0μg/rabbit; 6-7 μg/kg) in 0.2 mL volume doses. Adult rabbits were dosedwith Tc-99m labeled peptide intravenously in a lateral ear vein by slowbolus infusion (approximately 0.1 mL/min). A gamma camera fitted with apin-hole collimator (5 mm aperture) and energy window set for Tc-99m andprogrammed to accumulate 500,000 counts or scan for a desired time.Shortly before imaging, animals were anesthetized with a mixture ofketamine and xylazine (5:1, 1 mL/kg intramuscularly).

Gamma camera images were collected at 40-45° just above the heart (leftanterior oblique [LAO] view) to delineate the aortic arch and view thedescending aorta. Images were acquired at 1 and 2 h and occasionally at3 and 5 h after injection. Supplementary anesthesia was injected asneeded prior to each image collection.

At 2.5 h (after a 2 h scan), animals were sacrificed with an intravenousdose of sodium pentobarbital. Upon necropsy, the aorta was removed andbranching vessels dissected free from the aortic valve to themid-abdominal region. Using a parallel hole collimator, the aorta wasimaged ex corpora. Next, the aortae were opened longitudinally andstained with Sudan IV, thereby turning atherosclerotic plaque a deep redbrick color. Lipid-free and uninjured aortic endothelium retains itsnormal, glistening white-pink appearance under these conditions.

The results of these experiments are shown in FIGS. 1-3. Both groups ofrabbits showed rapid systemic clearance of Tc-99m P215. Thescintigraphic images indicate that the hepatobiliary system provides theprincipal clearance pathway. Control (plaque-free) aortae were onlyvisible for a short time after injection, resulting from circulating,blood-borne radioactivity. Each of the HC-fed NZW rabbit aortae showed aunique pattern and intensity of plaque distribution when imaged excorpora. All the HC aortae had variable amounts of radioactivityaccumulation but were consistent in their display of the greatestdeposition in the region of the aortic arch, with lesser degrees ofaccumulation in the distal and proximal segments of the aorta.

Positive correlations were observed among the in vivo and ex corporaTc-99m P215 images and the deposition patterns of Sudan IV in theHC-treated rabbit aortae. In contrast, no control aortae showed anyregional uptake of labeled peptide. FIG. 1 shows the deposition patternof Sudan IV in 1 HC-treated and 4 control rabbit aortae. The dark areasindicate the location of atherosclerotic plaque. FIGS. 2 and 3 show thecorresponding in vivo and ex corpora images, respectively.

These results demonstrate that Tc-99m labeled P215 is capable of imagingatherosclerotic plaque in an animal with high uptake and rapidclearance, facilitating early observation. Additionally, normal aortictissue shows minimal uptake of labeled P215, thereby reducing thelikelihood of artifactual positive scintigraphic images.

EXAMPLE 5 In Vivo Imaging using Tc-99m Labeled Compound P357 of DeepVein Thrombosis in a Canine Model

Mongrel dogs (25-35 lb., fasted overnight) were sedated with acombination of ketamine and aceprozamine intramuscularly and thenanesthetized with sodium pentobarbital intravenously. An 18-gaugeangiocath was inserted in the distal half of the right femoral vein andan 8 mm Dacron®-entwined stainless steel embolization coil (Cook Co.,Bloomington Ind.) was placed in the femoral vein at approximatelymid-femur in each animal. The catheter was removed, the wound suturedand the placement of the coil documented by X-ray. The animals were thenallowed to recover overnight.

One day following coil placement, each animal was re-anesthetized,intravenous saline drips placed in each foreleg and a urinary bladdercatheter inserted to collect urine. The animal was placed supine under agamma camera which was equipped with a low-energy, all purposecollimator and photopeaked for Tc-99m. Images were acquired on a NucLearMac computer system.

Tc-99m labeled P357 [(CH₂ CO--Y_(D).Apc.GDCGGC_(Acm) GC_(Acm)GGC.amide)₂ -[BAT-BS]] [185-370 MBq (5-10 mCi) Tc-99m and 0.2-0.4 mgP357] was injected into one foreleg intravenous line at its point ofinsertion. The second line was maintained for blood collection. Anteriorimages over the legs were acquired for 500,000 counts or 20 min(whichever was shorter), at approximately 10-20 min, and atapproximately 1, 2, 3 and 4 h post-injection. Following the collectionof the final image, each animal was deeply anesthetized withpentobarbital. Two blood samples were collected on a cardiac punctureusing a heparinized syringe followed by a euthanazing dose of saturatedpotassium chloride solution administered by intercardiac or bolusintravenous injection. The femoral vein containing the thrombus andsamples of thigh muscle were then carefully dissected out. The thrombuswas then dissected free of the vessel and placed in a pre-weighed testtube. The thrombus samples were then weighed and counted in a gamma wellcounter in the Tc-99m channel. Known fractions of the injected doseswere counted as well.

Fresh thrombus weight, percent injected dose (%ID)/g in the thrombus andblood obtained just prior to euthanasia and thrombus/blood andthrombus/muscle ratios determined. Thrombus/background ratios weredetermined by analysis of the counts/pixel measured inregions-of-interest (ROI) drawn over the thrombus and adjacent musclefrom computer-stored images.

Deep vein thrombus imaging was studied in a total of eight dogs. Tissuedata from these experiments are shown in the following Table.Representative examples of images acquired in an anterior view over thehind legs of one dog at 23, 71, 139, 208 and 222 min is presented inFIG. 4. These images show indications of labeled peptide uptake as earlyas 23 min post-injection, with unequivocal localization by 71 min whichpersisted until the end of the imaging period.

These results demonstrate that deep vein thrombi can be rapidly andefficiently located in vivo. Localization was clearly established within1 h post-injection and persisted, with increasing contrast and focaldefinition, over nearly 4 h post-injection.

    ______________________________________                                              Thrombus/ % ID/g     Thrombus/                                          IC.sub.50                                                                           Background                                                                              Thrombus   Blood   Thrombus/Muscle                            ______________________________________                                        0.081.sup.a                                                                         2.3.sup.a 0.016 ± 0.005                                                                         5.6 ± 1.4                                                                          17 ± 4.7                                ______________________________________                                         Values shown are the average ± the standard deviation from the mean;       [.sup.a =n = 1                                                           

The IC₅₀ value shown in the Table was determined as follows. Plateletaggregation studies were performed essentially as described by Zucker(1989, Methods in Enzymol. 169: 117-133). Briefly, platelet aggregationwas assayed with or without putative platelet aggregation inhibitorycompounds using fresh human platelet-rich plasma, comprising 300,000platelets per microliter. Platelet aggregation was induced by theaddition of a solution of adenosine diphosphate to a final concentrationof 10 to 15 micromolar, and the extent of platelet aggregation monitoredusing a Bio/Data aggregometer (Bio/Data Corp., Horsham, Pa.). Theconcentrations of platelet aggregation inhibitory compounds used werevaried from 0.1 to 500 μg/mL. The concentration of inhibitor thatreduced the extent of platelet aggregation by 50% (defined as the IC₅₀)was determined from plots of inhibitor concentration versus extent ofplatelet aggregation. An inhibition curve for peptide RGDS wasdetermined for each batch of platelets tested as a positive control.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 16                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 -      Arg Ala Leu Val Asp Thr Leu Lys - # Phe Val Thr Gln Ala Glu Gly        Ala                                                                           #   15                                                                        -      Lys                                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 -      Arg Ala Leu Val Asp Thr Glu Phe - # Lys Val Lys Gln Glu Ala Gly        Ala                                                                           #   15                                                                        -      Lys                                                                    - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 18 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 -      Pro Leu Ala Arg Ile Thr Leu Pro - # Asp Phe Arg Leu Pro Glu Ile        Ala                                                                           #   15                                                                        -      Ile Pro                                                                - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 -      Gly Gln Gln His His Leu Gly Gly - # Ala Lys Ala Gly Asp Val            #   10                                                                        - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 -      Pro Leu Tyr Lys Lys Ile Ile Lys - # Lys Leu Leu Glu Ser                #   10                                                                        - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 -      Leu Arg Ala Leu Val Asp Thr Leu - # Lys                                #  5 1                                                                        - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 -      Gly Gly Gly Leu Arg Ala Leu Val - # Asp Thr Leu Lys                    #   10                                                                        - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 -      Gly Gly Gly Leu Arg Ala Leu Val - # Asp Thr Leu Lys Phe Val Thr        Gln                                                                           #   15                                                                        -      Ala Glu Gly Ala Lys                                                                     20                                                           - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 -      Gly Gly Gly Arg Ala Leu Val Asp - # Thr Leu Lys Ala Leu Val Asp        Thr                                                                           #   15                                                                        -      Leu                                                                    - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 28 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                -      Gly His Arg Pro Leu Asp Lys Lys - # Arg Glu Glu Ala Pro Ser Leu        Arg                                                                           #   15                                                                        -      Pro Ala Pro Pro Pro Ile Ser Gly - # Gly Gly Tyr Arg                    #                 25                                                          - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                -      Pro Ser Pro Ser Pro Ile His Pro - # Ala His His Lys Arg Asp Arg        Arg                                                                           #   15                                                                        -      Gln                                                                    - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4..7                                                  #/label= Variantaas INFORMATION:                                              #"The phenylalanine and tryptophan residues                                   #D stereoisomers, and residue X is                                                           L-(4-chlorop - #henyl) alanine"                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                -      Gly Gly Gly Phe Xaa Tyr Trp Lys - # Thr Phe Thr                        #   10                                                                        - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 3..9                                                  #/label= VariantHER INFORMATION:                                              #"Residue X is L-[S-(3-aminopropyl)                                                          cysteine"                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                -      Gly Gly Cys Asn Pro Xaa Gly Asp - # Cys                                #  5 1                                                                        - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                -      Gly Gly Gly Leu Arg Ala Leu Val - # Asp Thr Leu Lys                    #   10                                                                        - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                -      Gly Cys Gly Gly Gly Leu Arg Ala - # Leu Val Asp Thr Leu Lys            #   10                                                                        - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                -      Gly Cys Tyr Arg Ala Leu Val Asp - # Thr Leu Lys Phe Val Thr Gln        Ala                                                                           #   15                                                                        -      Glu Gly Ala Lys                                                                         20                                                           __________________________________________________________________________

What is claimed is:
 1. A reagent for preparing a scintigraphic imagingagent, said reagent comprising a specific binding peptide having fromabout three to about 100 amino acids and being covalently linked to aradiolabel-binding moiety that forms an electrically neutral complexwhen bound to a radioisotope.
 2. The reagent of claim 1, wherein thepeptide and the moiety are covalently linked through from about one toabout 20 amino acids.
 3. The reagent of claim 1 wherein the radioisotopeis technetium-99m.
 4. The reagent of claim 1 wherein the peptide isselected from the group consisting of peptides having the amino acidsequences:formyl-MLF (VGVAPG)₃ amide (VPGVG)₄ amideRALVDTLKFVTQAEGAKamide (SEQ ID No.:1) RALVDTEFKVKQEAGAKamide (SEQ IDNo.:2) PLARITLPDFRLPEIAIPamide (SEQ ID No.:3) GQQHHLGGAKAGDV (SEQ IDNo.:4) PLYKKIIKKLLES (SEQ ID No.:5) LRALVDTLKamide (SEQ ID No.:6)GGGLRALVDTLKamide (SEQ ID No.:7) GGGLRALVDTLKFVTQAEGAKamide (SEQ IDNo.:8) GGGRALVDTLKALVDTLamide (SEQ ID No.:9)GHRPLDKKREEAPSLRPAPPPISGGGYR (SEQ ID No.:10) PSPSPIHPAHHKRDRRQamide (SEQID No.:11) GGGF_(D).Cpa.YW_(D) KTFTamide (SEQ ID No.:12) ##STR6##(SYNRGDSTC)₃ -TSEA GGGLRALVDTLKamide (SEQ ID No.:14) GCGGGLRALVDTLKamide(SEQ ID No.:15) GCYRALVDTLKFVTQAEGAKamide (SEQ ID No.:16) andGC(VGVAPG)₃ amide.
 5. A reagent for preparing a scintigraphic imagingagent comprising:a) at least two specific binding peptides, each havingan amino acid sequence of from about1 three to about 100 amino acids; b)at least one radiolabel binding moiety capable of forming anelectrically neutral complex with a radioisotope; and c) a polyvalentlinking moiety covalently linked to the peptides and to the radiolabelbinding moiety, thereby forming a multimer,wherein the molecular weightof the reagent is less than about 20,000 daltons.
 6. The reagent ofclaim 5 wherein the polyvalent linking moiety isbis-succinimidylmethylether, 4-(2,2-dimethylacetyl)benzoic acid,N-[2-(N',N'-bis(2-succinimidoethyl)aminoethyl)]-N⁶,N⁹-bis(2-methyl-2-mercaptopropyl)-6,9-diazanonanamide,tris(succinimidylethyl)amine or a derivative thereof.
 7. A scintigraphicimaging agent comprising the reagent of claim 1, wherein the moiety isbound to a radiolabel.
 8. The agent of claim 7, wherein the radiolabelis technetium-99m.
 9. A process of preparing the reagent of claim 1,wherein the peptide is chemically synthesized in vitro.
 10. The processof claim 9, wherein the peptide is synthesized by solid phase peptidesynthesis.
 11. A reagent for preparing a scintigraphic imaging agent,said reagent comprising a specific binding peptide having from about 3to about 100 amino acids and being covalently linked to aradiolabel-binding moiety of formula: ##STR7## wherein X=H or aprotecting group;(amino acid)=any amino acid;and wherein the moietyforms an electrically neutral complex when bound to a radioisotope. 12.The reagent of claim 11, wherein (amino acid) is glycine and X is anacetamidomethyl protecting group.
 13. The reagent of claim 11, whereinthe peptide and the moiety are covalently linked through from about oneto about 20 amino acids.
 14. The reagent of claim 11 wherein theradioisotope is technetium-99m.
 15. The reagent of claim 11, wherein thepeptide is selected from the group consisting of:formyl-MLF (VGVAPG)₃amide (VPGVG)₄ amide RALVDTLKFVTQAEGAKamide (SEQ ID No.:1)RALVDTEFKVKQEAGAKamide (SEQ ID No.:2) PLARITLPDFRLPEIAIPamide (SEQ IDNo.:3) GQQHHLGGAKAGDV (SEQ ID No.:4) PLYKKIIKKLLES (SEQ ID No.:5)LRALVDTLKamide (SEQ ID No.:6) GGGLRALVDTLKamide (SEQ ID No.:7)GGGLRALVDTLKFVTQAEGAKamide (SEQ ID No.:8) GGGRALVDTLKALVDTLamide (SEQ IDNo.:9) GHRPLDKKREEAPSLRPAPPPISGGGYR (SEQ ID No.:10)PSPSPIHPAHHKRDRRQamide (SEQ ID No.:11) GGGF_(D).Cpa.YW_(D) KTFTamide(SEQ ID No.:12) ##STR8## (SYNRGDSTC)₃ -TSEA GGGLRALVDTLKamide (SEQ IDNo.:14) GCGGGLRALVDTLKamide (SEQ ID No.:15) GCYRALVDTLKFVTQAEGAKamide(SEQ ID No.:16) and GC(VGVAPG)₃ amide.
 16. The reagent of claim 5,wherein the radiolabel-binding moiety is selected from the groupconsisting of: ##STR9## wherein X=H or a protecting group;(aminoacid)=any amino acid; ##STR10## wherein X=H or a protecting group;(amino acid)=any amino acid; ##STR11## wherein each R is independentlyH, CH₃ or C₂ H₅ ; each (pgp)^(S) is independently a thiol protectinggroup or H; m, n and p are independently 2 or 3; A=linear lower alkyl,cyclic lower alkyl, aryl, heterocyclyl, a combination thereof, or asubstituted derivative thereof; and ##STR12## wherein each R isindependently H, CH₃ or C₂ H₅ ; m, n and p are independently 2 or 3;A=linear lower alkyl, cyclic lower alkyl, aryl, heterocyclyl, acombination thereof, or a substituted derivative thereof; V=H or--CO-peptide; R'=H or peptide;and wherein when V=H, R'=peptide and whenR'=H, V=--CO-peptide.
 17. The reagent of claim 16, wherein thepolyvalent linking moiety is bis-succinimidylmethylether,4-(2,2-dimethylacetyl)benzoic acid,N-[2-(N',N'-bis(2-succinimido-ethyl)aminoethyl)]-N⁶,N⁹-bis(2-methyl-2-mercaptopropyl)-6,9-diazanonanamide,tris(succinimidylethyl)amine or a derivative thereof.
 18. Ascintigraphic imaging agent comprising the reagent of claim 11, whereinthe moiety is bound to a radiolabel.
 19. The agent of claim 18, whereinthe radiolabel is technetium-99m.
 20. A process of preparing the reagentof claim 11, wherein the peptide is chemically synthesized in vitro. 21.The process of claim 20, wherein the peptide is synthesized by solidphase peptide synthesis.
 22. A reagent for preparing an scintigraphicimaging agent, said reagent comprising a specific binding peptide havingfrom about 3 to about 100 amino acids, and being covalently linked to aradiolabel-binding moiety, wherein the moiety forms an electricallyneutral complex when bound to a radioisotope, said moiety having aformula selected from the group consisting of: ##STR13## wherein each Ris independently H, CH₃ or C₂ H₅ ;each (pgp)^(S) is independently athiol protecting group or H; m, n and p are independently 2 or 3;A=linear lower alkyl, cyclic lower alkyl, aryl, heterocyclyl, acombination thereof, or a substituted derivative thereof; and ##STR14##wherein each R is independently H, CH₃ or C₂ H₅ ; m, n and p areindependently 2 or 3; A=linear lower alkyl, cyclic lower alkyl, aryl,heterocyclyl, a combination thereof, or a substituted derivativethereof; V=H or --CO-peptide; R'=H or peptide;and wherein when V=H,R'=peptide and when R'=H, V=--CO-peptide.
 23. The reagent of claim 22,wherein the peptide and the moiety are covalently linked through fromabout one to about 20 amino acids.
 24. The reagent of claim 22, whereinthe radioisotope is technetium-99m.
 25. The reagent of claim 22, whereinthe peptide is selected from the group consisting of:formyl-MLF(VGVAPG)₃ amide (VPGVG)₄ amide RALVDTLKFVTQAEGAKamide (SEQ ID No.:1)RALVDTEFKVKQEAGAKamide (SEQ ID No.:2) PLARITLPDFRLPEIAIPamide (SEQ IDNo.:3) GQQHHLGGAKAGDV (SEQ ID No.:4) PLYKKIIKKLLES (SEQ ID No.:5)LRALVDTLKamide (SEQ ID No.:6) GGGLRALVDTLKamide (SEQ ID No.:7)GGGLRALVDTLKFVTQAEGAKamide (SEQ ID No.:8) GGGRALVDTLKALVDTLamide (SEQ IDNo.:9) GHRPLDKKREEAPSLRPAPPPISGGGYR (SEQ ID No.:10)PSPSPIHPAHHKRDRRQamide (SEQ ID No.:11) GGGF_(D).Cpa.YW_(D) KTFTamide(SEQ ID No.:12) ##STR15## (SYNRGDSTC)₃ -TSEA GGGLRALVDTLKamide (SEQ IDNo.:14) GCGGGLRALVDTLKamide (SEQ ID No.:15) GCYRALVDTLKFVTQAEGAKamide(SEQ ID No.:16) and GC(VGVAPG)₃ amide.
 26. A scintigraphic imaging agentcomprising the reagent of claim 22, wherein the moiety is bound to aradiolabel.
 27. The agent of claim 26, wherein the radiolabel istechnetium-99m.
 28. A process of preparing the reagent of claim 22,wherein the peptide is chemically synthesized in vitro.
 29. The processof claim 28, wherein the peptide is synthesized by solid phase peptidesynthesis.
 30. A composition comprising an ε-amino group of aN-α-protected lysine attached to a radiolabel-binding moiety having aformula selected from the group consisting of: ##STR16## wherein each Ris independently H, CH₃ or C₂ H₅ but if X=H, one R=Y;(pgp)_(N) =an amineprotecting group or H; each (pgp)^(S) is independently a thiolprotecting group or H; m, n and p are independently 2 or 3; X=H or--A--COOH, but if X=H, one R=Y and (pgp)_(N) is not H; Y=--A--COOH;A=linear lower alkyl, cyclic lower alkyl, aryl, heterocyclyl, acombination thereof, or a substituted derivative thereof; ##STR17##wherein each R is independently H, CH₃ or C₂ H₅ but if Z=H, one R=Y;each (pgp)_(N) is an amine protecting group or H; each (pgp)^(S) isindependently a thiol protecting group or H; m, n and p areindependently 2 or 3; Z=H or --A--CH(V)NH(pgp)₂ ^(N), but if Z=H, oneR=Y; Y=--A--CH(V)NH(pgp)₂ ^(N) ; A=linear lower alkyl, cyclic loweralkyl, aryl, heterocyclyl, a combination thereof, or a substitutedderivative thereof; V=H or COOH;and wherein if (pgp)₁ ^(N) and V are H,then (pgp)^(S) is not H and if (pgp)^(S) and V are H, then (pgp)^(N) isnot H, and if V is H, (pgp)₁ ^(N) is not H; and ##STR18## wherein each Ris independently H, CH₃ or C₂ H₅ and one R=Y; each (pgp)^(N) is an amineprotecting group or H; each (pgp)^(S) is independently a thiolprotecting group or H; m, n and p are independently 2 or 3;Y=--A--CH(V)NH(pgp)₂ ^(N) ; A=linear or cyclic lower alkyl, aryl,heterocyclyl, a combination thereof, or a substituted derivativethereof; V=H or COOH;and wherein if (pgp)₂ ^(N) and V are H, then(pgp)^(S) is not H and if (pgp)^(S) and V are H, then (pgp)₂ ^(N) is notH, and at least one (pgp)₁ ^(N) moiety is not H.
 31. A compositioncomprising [N-ε-(N⁹ -t-butoxycarbonyl)-N⁶,N⁹-bis[2-methyl-2-(triphenylmethylthio)propyl]-6,9-diazanonanoyl)-N-α-Fmoc-lysine.